Microbial diversity in lake sediments detected by PCR-DGGE

In this study, PCR-denaturing gradient gel electrophoresis (DGGE) was applied to analyze the microbial communities in lake sediments from Lake Xuanwu, Lake Mochou in Nanjing and Lake Taihu in Wuxi. Sediment samples from seven locations in three lakes were collected and their genomic DNAs were extracted. The DNA yields of the sediments of Lake Xuanwu and Lake Mochou were high (10 μg/g), while that of sediments in Lake Taihu was relatively low. After DNA purification, the 16S rDNA genes (V3 to V5 region) were amplified and the amplified DNA fragments were separated by parallel DGGE. The DGGE profiles showed that there were five common bands in all the lake sediment samples indicating that there were similarities among the populations of microorganisms in all the lake sediments. The DGGE profiles of Lake Xuanwu and Lake Mochou were similar and about 20 types of microorganisms were identified in the sediment samples of both lakes. These results suggest that the sediment samples of these two city lakes (Xuanwu, Mochou) have similar microbial communities. However, the DGGE profiles of sediment samples in Lake Taihu were significantly different from these two lakes. Furthermore, the DGGE profiles of sediment samples in different locations in Lake Taihu were also different, suggesting that the microbial communities in Lake Taihu are more diversified than those in Lake Xuanwu and Lake Mochou. The differences in microbial diversity may be caused by the different environmental conditions, such as redox potential, pH, and the concentrations of organic matters. Seven major bands of 16S rDNA genes fragments from the DGGE profiles of sediment samples were further re-amplified and sequenced. The results of sequencing analysis indicate that five sequences shared 99%–100% homology with known sequences (Bacillus and Brevibacillus, uncultured bacteria), while the other two sequences shared 93%–96% homology with known sequences (Acinetobacter, and Bacillus). The study shows that the PCR-DGGE technique combined with sequence analysis is a feasible and efficient method for the determination of microbial communities in sediment samples. __________ Translated from Acta Ecologica Sinica, 2006, 26(11): 3610–3616 [译自: 生态学报]     

Bacterial and archaeal assemblages in sediments of a large shallow freshwater lake, Lake Taihu, as revealed by denaturing gradient gel electrophoresis

Aims:  To explore the association of microbial community structure with the development of eutrophication in a large shallow freshwater lake, Lake Taihu.
Methods and Results:  The bacterial and archaeal assemblages in sediments of different lake areas were analysed using denaturing gradient gel electrophoresis (DGGE) of amplified 16S rDNA fragments. The bacterial DGGE profiles showed that eutrophied sites, grass-bottom areas and relatively clean sites with a eutrophic (albeit dredged) site are three respective clusters. Fifty-one dominant bacterial DGGE bands were detected and 92 corresponding clones were sequenced, most of which were affiliated with bacterial phylotypes commonly found in freshwater ecosystems. Actinobacteria were detected in the centre of the lake and not at eutrophied sites whereas the opposite was found with respect to Verrucomicrobiales . Twenty-five dominant archaeal DGGE bands were detected and 31 corresponding clones were sequenced, most of which were affiliated with freshwater archaeal phylotypes.
Conclusions:  The bacterial community structures in the sediments of different areas with similar water quality and situation tend to be similar in Taihu Lake.
Significance and Impact of the Study:  This study may expand our knowledge on the relationship between the overall microbial assemblages and the development of eutrophication in the shallow freshwater lake.     

洪湖沉积物中真菌群落结构的垂直分布

湖泊沉积物中真菌的类群及其作用所知甚少。以长江中上游代表性湖泊洪湖为研究对象,从湖心截取了一个72cm长的沉积柱,通过rDNA内转录间隔区（ITS）的变性梯度凝胶电泳（DGGE）,对洪湖沉积物中真菌群落结构的垂直变化进行了分析。结果显示,DGGE条带以14－18cm层次为过渡,在2－14cm和14－72cm之间存在一定差异。Shannon-Weaver index（H′）在上下层之间有波动。聚类分析上层2－18cm和中下层18－72cm分别以不同的小类聚在一起。表明14－18cm以上和以下的类群存在差异,     

Vertical distribution of bacterial community structure in the sediments of two eutrophic lakes revealed by denaturing gradient gel electrophoresis (DGGE) and multivariate analysis techniques

Vertical distribution of bacterial community structure was investigated in the sediments of two eutrophic lakes of China, Lake Taihu and Lake Xuanwu. Profiles of bacterial communities were generated using a molecular fingerprinting technique, denaturing gradient gel electrophoresis (DGGE) followed by DNA sequence analysis, and the results were interpreted with multivariate statistical analysis. To assess changes in the genetic diversity of bacterial communities with changing depth, DGGE banding patterns were analysed by cluster analysis. Distinct clusters were recognized in different sampling stations of Lake Taihu. Canonical correspondence analysis (CCA) was carried out to infer the relationship between environmental variables and bacterial community structure. DGGE samples collected at the same sampling site clustered together in both lakes. Total phosphorus, organic matter and pH were considered to be the key factors driving the changes in bacterial community composition.     

Genetic diversity of picoeukaryotes in eight lakes differing in trophic status

The genetic diversity of picoeukaryotes (0.2-5.0 μm) was investigated in 8 lakes differing in trophic status in Nanjing, China. Denaturing gradient gel electrophoresis (DGGE) and cloning and sequencing of 18S rRNA genes were applied to analyze the picoeukaryotic communities. DGGE analysis showed that among the 8 lakes, the diversity of picoeukaryotes was greatest in the mesotrophic Lake Nan (24 bands) and least in the oligotrophic Lake Qian (12 bands). Cluster analysis of DGGE profiles revealed that the 8 lakes were grouped into 2 distinct clusters. Cluster 1 contained lakes Mochou, Zixia, Huashen, Nan, Pipa, and Qian, while cluster 2 contained lakes Xuanwu and Baijia. Clone libraries were constructed from the mesotrophic Lake Xuanwu and the oligotrophic Lake Zixia, and the 2 libraries were compared using the program LIBSHUFF. This analysis indicated that the picoeukaryotic community composition differed significantly between the 2 lakes (p = 0.001). A total of 25 operational taxonomic units were detected; 18 (62 clones) were related to known eukaryotic groups, while 7 (30 clones) were not affiliated with any known eukaryotic group. Alveolates and stramenopiles were the dominant groups in Lake Xuanwu, while alveolates and chlorophyta predominated in Lake Zixia. Multivariate statistical analysis indicated that the differences in the picoeukaryotic community composition of the 8 lakes might be related to trophic status and top-down regulation by metazooplankton.     

Diversity of Kenyan soda lake alkaliphiles assessed by molecular methods

DNA was extracted from water and sediment samples taken from soda lakes of the Kenyan-Tanzanian Rift Valley. DNA was also extracted from microbial enrichment cultures of sediment samples. 16S rRNA genes were amplified by the polymerase chain reaction and microbial diversity was studied using denaturing gradient gel electrophoresis (DGGE) of 16S rDNA amplicons. Cloning and sequencing of single DGGE bands showed that they usually contained mixed amplicons. Several of the amplicon sequences had high identities, up to 99%, with 16S rRNA genes of organisms previously isolated from soda lakes, while others were only distantly related, with identities as low as 82%. Phylogenetic analysis of the sequenced amplicons indicated that sequences were related to the haloarchaeal, Bacillus/Clostridium, Rhodobacterium/Thioalcalovibrio/ Methylobacter, and Cytophaga/Flavobacterium/Bacteroides (CFB) groups and the enterobacteria/Aeromonas/Vibrio part of the 3 subdivision of the Proteobacteria.Communicated by K. Horikoshi     

应用DGGE法对青海相邻两盐湖中细菌多样性的快速检测

分别对青海相邻两盐湖柯柯盐湖、茶卡盐湖土样、泥样进行富集培养后,从中提取的DNA用两套不同的细菌通用引物进行扩增,分别得到包含V8和V9高变区的16SrDNA片断。经变性梯度凝胶电泳(DGGE)分析,结果显示这两个盐湖富集样品中细菌多样性具有较大的差异,而且同一盐湖不同性质样品中的细菌多样性差异也较大,两湖的泥样富集样品中均表现出了稍丰富的多样性。     

The microbial composition of three limnologically disparate hypersaline Antarctic lakes

16S rRNA clone library analysis was used to examine the biodiversity and community structure within the sediments of three hypersaline Antarctic lakes. Compared to sediment of low to moderate salinity Antarctic lakes the species richness of the hypersaline lake sediments was 2-20 times lower. The community of Deep Lake (32% salinity, average sediment temperature -15 degrees C) was made up almost entirely of halophilic Archaea. The sediment communities of two meromictic hypersaline lakes, Organic Lake (20% salinity, -7 degrees C) and Ekho Lake (15% salinity, 15 degrees C) were more complex, containing phylotypes clustering within the Proteobacteria and Cytophagales divisions and with algal chloroplasts. Many phylotypes of these lakes were related to taxa more adapted to marine-like salinity and perhaps derive from bacteria exported into the sediment from the lower salinity surface waters. The Ekho Lake clone library contained several major phylotypes related to the Haloanaerobiales, the growth of which appears to be promoted by the comparatively high in situ temperature of this lake.     

有机污染物对水体真细菌群落结构的影响

为了揭示有机污染物对环境真细菌组成和多样性的影响,应用末端限制性片段长度多态性(tRFLP)和16S rDNA文库技术并结合水质分析方法,比较分析了松花江流域内受不同程度有机污染的4个水体及其沉积物中真细菌的群落结构。tRFLP分析表明各水体及底泥均呈现较为复杂的群落结构模式,不同底泥群落形成的末端限制性片段(TRF)图谱具有很高的相似性,但随着污染程度的加强,部分类群明显富集,而且在水样组和泥样组内,群落结构的相似性同水质相似性是一致的,主成分分析(PCA)显示水样和泥样中的真细菌TRF形成不同的群。16SrDNA文库分析表明松花江哈尔滨段底泥中真细菌分布于10个门,Proteobacteria门占优势,达群落总数的21.92%(β-Proteobacteria亚门占10.96%),而有机染污物严重超标的生活污水排污道底泥中的微生物多样性较低,分布于7个门,Proteobacteria门为优势群,占群落的47.37%(α-Proteobacteria亚门占21.05%,δ/ε-Proteobacteria亚门占15.79%)。该研究表明向水体中长期排放高浓度有机物能使系统中微生物群落多样性降低,与污染物降解相关的功能微生物类群明显富集。     

Characterization of the depth-related changes in the microbial communities in Lake Hovsgol sediment by 16S rRNA gene-based approaches

The undisturbed sediment of Lake Hovsgol (Mongolia) is scientifically important because it represents a record of the environmental changes that took place between the Holocene (the present age) and Pleistocene (the last ice age; 12,000 14C years before present day). Here, we investigated how the current microbial communities change as the depth increases by PCR-denaturing gradient gel electrophoresis (DGGE) analysis of the 16S rRNA genes of the microbial communities. The microbial diversity, as estimated by the Shannon index, decreased as the depth increased. In particular, significant changes in archaeal diversity were observed in the middle depth (at 39-42 cm depth of total 60 cm depth) that marks the border between the Holocene and Pleistocene. Phylotype belonging to Beta-and Gamma-Proteobacteria were the predominant bacteria and most of these persisted throughout the depth examined. However, as the depth increased, some bacteria (some genera belonging to Beta-Proteobacteria, Nitrospira, and OP8-9) were not detectable while others (some genera belonging to Alpha-, Beta-, Gamma-Proteobacteria) newly detected by DGGE. Crenarchaea were the predominant archaea and only one phylotype belonging to Euryarchaea was found. Both the archaeal and bacterial profiles revealed by the DGGE band patterns could be grouped into four and three subsets, respectively, subsets that were largely divided by the border between the Holocene and Pleistocene. Thus, the diversity of the current microbial communities in Lake Hovsgol sediments decreases with increasing depth. These changes probably relate to the environmental conditions in the sediments, which were shaped by the paleoclimatic events taking place between the Holocene and Pleistocene.     

Bacterial Diversity among the Sediments of Glacial Lakes in the Western Balkans: Exploring the Impact of Human Population

16S rRNA gene-based metagenomic approach was used to assess the biodiversity of bacterial communities in the sediments of selected glacial lakes in the Western Balkans and to assess the impact of human population on these microbial communities. Sediment samples were collected from three glacial lakes, viz., Plav Lake (in a zone of the highest impact of human population), Black Lake (a zone of medium impact of human population), and Donje Bare Lake (a remote lake with minimal impact of human population).

Canonical correlation analysis analysis indicated correlation between the distance of the lake from urbanized population and bacterial diversity in Donje Bare Lake sediment. Bacterial diversity of Black Lake sediment was correlated with high content of phosphorous and pH value. Chemical compounds exhibiting the most prominent correlation with bacterial diversity of Plav Lake were NH₄-N, K₂O, CaCo₃, and total nitrogen . Additionally, CCA analysis indicated that population density was correlated with biodiversity of bacterial communities in Plav Lake sediment, which is the most exposed to human population. Multivariate regression revealed the highest correlation between the presence of Proteobacteria classes and population density and levels of NH₄-N.

The influence of human population was observed to be important for shaping the sediment communities in addition to biological and chemical factors.     

Metabolic and molecular characterization of bacterial community associated to Patagonian Chilean oligotrophic-lakes of quaternary glacial origin

The Patagonian Lakes have particular environmental conditions with or without intermittent disturbances. The study of the microorganisms present in aquatic ecosystems has increased notably because they can be used as micro-scale bioindicators of, among others, anthropogenic pollution and climatic change. The aim of the work was to compare the composition of the bacterial communities associated with sediments of three Patagonian Lakes with different geomorphologic patterns and disturbances. The lake sediments were characterized by molecular techniques, physiology profiles and physico-chemical analyses. The metabolic and physiological profiles of the microbial community demonstrated that non-impacted Tranquilo Lake is statistically different to impacted Bertrand and Plomo Lakes. Similar results were detected by DGGE profiles. FISH results demonstrated that betaproteobacteria showed the highest count in the Tranquilo Lake while gammaproteobacteria showed the highest counts in the Bertrand and Plomo Lakes, indicating that their sediments are highly dystrophic. The results demonstrate differences in the metabolic activity and structural and functional composition of bacterial communities of the studied lakes, which have different geomorphological patterns due to disturbances such as volcanic activity and the climatic change.     

PCR指纹图谱技术分析人体口腔内微生物菌群结构

目的 应用PCR-DGGE指纹图谱技术对人体口腔微生物菌群结构进行系统性研究.方法 对1例健康人唾液周期性采集的样品和8例健康人个体的唾液与牙菌斑采集的样品,进行微生物群落总DNA的抽提.以此为模板扩增16S rRNA V3可变区,产物经DGGE指纹图谱分析其组成结构,并运用UVIBAND/MAP等软件比较所得群落指纹图谱的相似性指数.结果 同一健康人个体不同采样时间的唾液菌群结构相似性系数＞74%,通过对不同健康个体口腔样本的研究,发现同一个体的唾液与牙菌斑菌群结构存在差异（84%～95%）.结论 同一健康个体其唾液微生物菌群在一定时间内基本稳定,仅有微小的变化;唾液与同个体牙菌斑的微生物组成虽然存在差异,但这种差异要明显小于个体间的差异.     

Diversity analysis of bacterial community compositions in sediments of urban lakes by terminal restriction fragment length polymorphism (T-RFLP)

Bacteria are crucial components in lake sediments and play important role in various environmental processes. Urban lakes in the densely populated cities are often small, shallow, highly artificial and hypereutrophic compared to rural and natural lakes and have been overlooked for a long time. In the present study, bacterial community compositions in surface sediments of three urban lakes (Lake Mochou, Lake Qianhu and Lake Zixia) in Nanjing City, China, were investigated using the terminal restriction fragment length polymorphism (T-RFLP) of PCR-amplified 16S rRNA gene and clone libraries. Remarkable differences in the T-RFLP patterns were observed in different lakes or different sampling stations of the same lake. Canonical correspondence analysis indicated that total nitrogen (TN) had significant effects on bacterial community structure in the lake sediments. Chloroflexi were the most dominant bacterial group in the clone library from Lake Mochou (21.7?% of the total clones) which was partly associated with its higher TN and organic matters concentrations. However, Bacteroidetes appeared to be dominated colonizers in the sediments of Lake Zixia (20.4?% of the total clones). Our study gives a comprehensive insight into the structure of bacterial community of urban lake sediments, indicating that the environmental factors played a key role in influencing the bacterial community composition in the freshwater ecosystems.     

PCR-DGGE技术在农田土壤微生物多样性研究中的应用

变性梯度凝胶电泳技术(DGGE)在微生物生态学领域有着广泛的应用。研究采用化学裂解法直接提取出不同农田土壤微生物基因组DNA,并以此基因组DNA为模板,选择特异性引物F357GC和R515对16S rRNA基因的V3区进行扩增,长约230bp的PCR产物经变性梯度凝胶电泳(DGGE)进行分离后,得到不同数目且分离效果较好的电泳条带。结果说明,DGGE能够对土壤样品中的不同微生物的16S rRNA基因的V3区的DNA扩增片断进行分离,为这些DNA片断的定性和鉴定提供了条件。与传统的平板培养方法相比,变性梯度凝胶电泳(DGGE)技术能够更精确的反映出土壤微生物多样性,它是一种有效的微生物多样性研究技术。     

Eutrophication as a driver of microbial community structure in lake sediments

Lake sediments are globally important carbon sinks. Although the fate of organic carbon in lake sediments depends significantly on microorganisms, only few studies have investigated controls on lake sedimentary microbial communities. Here we investigate the impact of anthropogenic eutrophication, which affects redox chemistry and organic matter (OM) sources in sediments, on microbial communities across five lakes in central Switzerland. Lipid biomarkers and distributions of microbial respiration reactions indicate strong increases in aquatic OM contributions and microbial activity with increasing trophic state. Across all lakes, 16S rRNA genes analyses indicate similar depth-dependent zonations at the phylum- and class-level that follow vertical distributions of OM sources and respiration reactions. Yet, there are notable differences, such as higher abundances of nitrifying Bacteria and Archaea in an oligotrophic lake. Furthermore, analyses at the order-level and below suggest that changes in OM sources due to eutrophication cause permanent changes in bacterial community structure. By contrast, archaeal communities are differentiated according to trophic state in recently deposited layers, but converge in older sediments deposited under different trophic regimes. Our study indicates an important role for trophic state in driving lacustrine sediment microbial communities and reveals fundamental differences in the temporal responses of sediment Bacteria and Archaea to eutrophication.     

Molecular Detection of Novel Anammox Bacterial Clusters in the Sediments of the Shallow Freshwater Lake Taihu

Previous studies have demonstrated the wide occurrence of anaerobic ammonium oxidizing (anammox) bacteria; however, there is very limited information on the distribution of these bacteria in freshwater habitats. In this study, the anammox bacterial communities were detected by molecular analysis targeting the 16S rRNA genes in the sediments of Lake Taihu, a large and shallow eutrophic freshwater lake in China. The recovery of specific 16S rRNA sequences with two stable monophyletic clusters indicated that anammox bacteria were present in Lake Taihu. A phylogenetic analysis indicated that these two groups represent two novel lineages within the first subgroup of anammox bacteria, independent of the treeing methods. High intra-lake variability in anammox bacterial diversity and community composition was observed, in particular, based on a 1% cut-off of 16S rRNA sequence variation. The spatial variability was largely related to the substrate availability, which was denoted by the correlations between the relative abundance of the two Taihu anammox bacterial groups and the concentrations of ammonium and nitrite. This indicates that the niche differentiation of anammox bacteria is linked to the environmental heterogeneity. These findings suggest that the freshwater lakes may accommodate different anammox bacterial communities and, thus, expand our knowledge on the diversity and distribution of anammox bacteria. Supplemental materials are available for this article. Go to the publisher's online edition of Geomicrobiology Journal to view the free supplemental files.     

Denaturing gradient gel electrophoresis analysis with different primers of subgingival bacterial communities under mechanical debridement

DGGE of 16S rDNA is one of the most frequently used methods to study microbial communities. In this study, the DGGE profiles of different 16S rDNA regions of the periodontal pathogens Porphyromonas gingivalis, Fusobacterium nucleatum, and Prevotella nigrescens were investigated. The results suggested that V3-V5 and V6-V8 fragments may be suitable for community analysis of subgingival bacteria. Further analysis of subgingival samples with V3-V5 and V6-V8 regions as target fragments suggested that, in chronic periodontitis, re-colonization by periodontal bacteria with a population very similar to the baseline may occur by 6 weeks after mechanical debridement.     

Community Structure of Bacteria Associated with Sheaths of Freshwater and Brackish Thioploca Species

Bacterial communities associated with sheaths of Thioploca spp. from two freshwater lakes (Lake Biwa, Japan, and Lake Constance, Germany) and one brackish lake (Lake Ogawara, Japan) were analyzed with denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene fragments. The comparison between the DGGE band patterns of bulk sediment and Thioploca filaments of Lake Biwa suggested the presence of specific bacterial communities associated with Thioploca sheaths. As members of sheath-associated communities, bacteria belonging to Bacteroidetes were detected from the samples of both freshwater lakes. A DGGE band from Thioploca of Lake Biwa, belonging to candidate division OP8, was quite closely related to another DGGE band detected from that of Lake Constance. In contrast to the case of freshwater lakes, no bacterium of Bacteroidetes or OP8 was detected from Thioploca of Lake Ogawara. However, two DGGE bands from Lake Ogawara, belonging to Chloroflexi, were quite closely related to a DGGE band from Lake Constance. Two DGGE bands obtained from Lake Biwa were closely related to phylogenetically distant dissimilatory Fe(III)-reducing bacteria. Cloning analyses for a dissimilatory sulfite reductase gene were performed on the same samples used for DGGE analysis. The results of the analyses suggest that sheaths of freshwater/brackish Thioploca have little ecological significance for the majority of sulfate reducers.     

Development of Temporal Temperature Gradient Electrophoresis for Characterising Methanogen Diversity

Temporal temperature gradient electrophoretic (TTGE) analysis of 16S rDNA sequences was optimized to monitor the methanogen population present in water and sediments of a small eutrophic lake, Priest Pot, in the English Lake district. The production of nonrepresentative TTGE profiles due to the generation of polymerase chain reaction (PCR) artifacts initially proved problematical. The use of a proofreading polymerase in the PCR was found to be essential and fully optimized protocols were established and tested to ensure confidence that the TTGE profiles truly reflected sequence diversity. TTGE analysis revealed the methanogen population to be less diverse in water than in sediment. The most genetic diversity was observed in TTGE profiles of sediment DNA isolated in winter and the least was in sediment DNA isolated in summer. DNA sequencing analysis of bands recovered from TTGE gels revealed the presence of two methanogen communities. One clustered with Methanosaeta species and the other with the Methanomicrobiales. Many sequences showed low DNA sequence similarity to known methanogens, suggesting that Priest Pot harbors previously undescribed methanogen species.